Genetic engineering techniques are essential for analysis of gene functions and production of useful proteins. In the past, production of useful proteins had usually used E. coli or yeast as hosts; however, such hosts had serious problems in terms of difficulty in mass-production. In recent years, accordingly, the silkworm (Bombyx mori) that enables synthesis of large quantities of proteins within a short period of time is hitting the limelight as a protein mass-production system. When silkworm is used as a host, a technique for producing a transformant into which a foreign gene has been introduced in its cell; that is, transgenic silkworm, becomes critical. A technique of stably maintaining a foreign gene in the genome of Bombyx mori with the use of the piggyBac transposon has been established (Tamura, T. et al., 2000, Nat. Biotechnol., 18: 81-84).
In addition to a technique of preparing a transformant, genetic engineering techniques require a technique of accurately and simply discriminating a transformant that carries an introduced foreign gene from a host that does not carry such foreign gene (i.e., a non-transformant). When hosts are insects, objective genes are introduced into the host cells with marker genes such as fluorescent proteins and promoter that controls the expression thereof at the time of transformation, and whether or not the host cells have been transformed is determined based on the expression of marker genes. When implementing such technique, accordingly, it is critical to select a promoter that can strongly and extensively induce the expression of marker genes.
In the case of silkworm, a systemic actin A3 gene promoter (Non-patent Literatures 1 and 2), the eye-specific 3×P3 promoter known to be versatile among various organism species (Non-patent Literature 3), and the immediate-early IE1 promoter (Non-patent Literature 4) have been primarily used as the promoters described above. However, these promoters had problems described below.
First of all, activity of the A3 promoter is weak at the embryonic stage, and a transformant cannot be discriminated at an early developmental stage, as shown in FIG. 1B. Accordingly, all the individuals that had been subjected to transformation had to be uniformly grown up to the larval stage at which expression of marker genes became identifiable. Therefore, the significant amounts of useless efforts and labors were necessitated and wasted expense accrued. In addition, larvae move around. Accordingly, when a phonotype based on a marker gene cannot be visually distinguished under visible light, unlike a body color, it was difficult to select individuals of interest. When a marker gene encodes a fluorescent protein, for example, a fluorescence-emitting individual needs to be selected while continuously applying an excitation light to silkworm larvae that move around.
Subsequently, the 3×P3 promoter already exhibits activity at the embryonic stage, and the problem of the A3 promoter thus does not arise. As shown in FIG. 1D, however, the site of expression is limited to a very small portion of an embryo (a portion indicated with an arrow in the figure). Accordingly, the technical skills to identify and determine the individuals of interest were required. When an expression vector carrying such promoter is inserted into a host genome, expression of the objective gene may be inhibited depending on the effect of an insertion site. In such a case, discrimination of the transformants became difficult for even a person skilled in the art. In addition, the duration during which the objective gene expression could be confirmed at the early developmental stage was as short as 1 or 2 days, disadvantageously.
In the case of IE1 promoter, activity of expression induction was not observed in the fat body, spermary, or ovary. This indicates that activity of gene expression induction was not effective throughout the body.